Influenza A viruses possess two surface spike proteins, hemagglutinin (HA) and neuraminidase (NA) (Lamb et al., 1996). The HA protein, a trimeric type I membrane protein, is responsible for binding to sialyloligosaccharides (oligosaccharides containing terminal sialic acid linked to galactose) on host cell surface glycoproteins or glycolipids (reviewed Wiley et al., 1987). This protein is also responsible for fusion between viral and host cell membranes, following virion internalization by endocytosis. Neuraminidase (NA), a tetrameric type II membrane protein, is a sialidase that cleaves terminal sialic acid residues from the glycoconjugates of host cells and the HA and NA, and thus is recognized as receptor-destroying enzyme (Air et al., 1989). This sialidase activity is necessary for efficient release of progeny virions from the host cell surface, as well as prevention of progeny aggregation due to the binding activity of viral HAs with other glycoproteins (Pause et al., 1974; Shibata et al., 1993). Thus, the receptor-binding activity of the HA and the receptor-destroying activity of the NA likely act as counterbalances, allowing efficient replication of influenza virus, e.g., influenza A virus.
Influenza A viruses of all known subtypes have been isolated from a variety of animals, including humans, wild and domestic birds, pigs, horses, and sea mammals (Webster et al., 1992). Viruses responsible for the 1957 and 1968 influenza pandemics were reassortants between human and avian viruses, with the PB1, HA and/or NA genes derived from the latter (Kawaoka et al., 1989; Laver et al., 1973; Scholtissek et al., 1978). Such interspecies transmission of avian virus genes forces adaptation of the gene products to the new environment (i.e., human respiratory organs).
Comparative studies have demonstrated that HA receptor specificity differs among influenza A viruses, depending on the animal species from which they were isolated (Rogers et al., 1983a; Rogers et al., 1983b). Thus, amino acids alterations are likely needed for efficient viral growth in new animal hosts. However, it is unclear if HA mutations alone, or in addition to mutations in HA, are needed for influenza virus adaptation in new hosts.
Thus, what is needed is a method to select for influenza virus mutants capable of replicating in an altered host cell.